Staphylococcus aureus is a versatile pathogen that causes a broad spectrum of acute and chronic infections. An important determinant of the chronic infections is the ability of S. aureus to develop a biofilm on host tissue or medical implant material. Biofilms are problematic for treatment due to their inherent resistance to antimicrobial therapies and host immune defenses, and these problems are compounded by the growing levels of methicillin-resistant S. aureus (MRSA). The long-term goal of my research program is to understand S. aureus biofilm development pathways in order to improve treatment approaches. Our studies have demonstrated that the quorum-sensing system, also called the accessory gene regulator or agr, is a key mediator of the biofilm lifestyle. For S. aureus to leave a biofilm and seed new sites, the agr system has to be reactivated to disassemble the biofilm structure, and once cells have dispersed, they regain susceptibility to antibiotic treatment. Recent studies in our group have identified a prominent role for agr-regulated extracellular proteases in this mechanism. Our central hypothesis is that S. aureus has an agr-regulated biofilm dispersal pathway that is mediated by cysteine proteases (called Staphopains). Our preliminary findings also demonstrate that biofilm inhibitory factors are produced by human neutrophils, leading us to hypothesize that host defenses can tap into the dispersal pathway and destroy biofilms. For Specific Aim 1, we hypothesize that agr and Rot regulation of Staphopain production controls biofilm dispersal. To evaluate this hypothesis and further define the dispersal mechanism, we will (i) investigate the contribution of Rot repressor to the agr-protease regulatory link through molecular approaches and biofilm assays; (ii) assess the role of Staphopains in biofilm maturation and dispersal; and (iii) test the conservation of the regulatory cascade in vivo using infection imaging in a murine catheter biofilm model. We developed a surface shaving proteomic method to identify Staphopain A cleavage targets. Using this method, we discovered that the Serine-Aspartate-Repeat (Sdr) proteins are removed by Staphopain A from the S. aureus surface in a biofilm state. We hypothesize that the Staphopains cleave the Sdr surface proteins to promote biofilm dispersal. To test this hypothesis, in Specific Aim 2 we will (i) perform an in-depth characterization of the function of Sdr proteins in biofilm development; (ii) biochemically define the mode of action of Staphopain A and B on the Sdr's; and (iii) determine the impact of glycosylation on Sdr protein processing and biofilm function. Finally, we discovered that S. aureus biofilms are hypersensitive to neutrophil granules (in collaboration with Dr. William Nauseef). Through purification, we identified the protease Cathepsin G as the primary anti-biofilm agent, and human neutrophil elastase (HNE) also showed activity. We hypothesize that neutrophil granule proteases inhibit biofilms by cleaving the Sdr proteins. To test this hypothesis, in Specific Aim 3 we will (i) perform neutrophil protease processing studies on the Sdr proteins; (ii) assess the impact of neutrophil pathway inhibition on anti-biofilm activities; ad (iii) identify and characterize Cathepsin G and HNE released proteins from the surface of biofilms. An improved understanding of biofilm dispersal mechanisms, and host modulation of these mechanisms, will aid the development of therapeutics that can provide innovative treatments for S. aureus chronic infections.